Overall Program Abstract. Dendritic spines with abnormal morphology occur in various forms of mental retardation and are also found in psychiatric conditions associated with disturbances to memory and cognition. These widely reported observations raise the possibility that defects in the processes that regulate the spine actin cytoskeleton are a common final substrate for a broad array of learning disabilities. The present proposal, which is a revised application for renewal of PPG #P01NS045260, addresses this hypothesis and potential therapeutic strategies suggested by it. Work under the previous PPG award showed that the stabilization of long-term potentiation (LTP), a form of synaptic plasticity closely related to the encoding of lasting memory, is moderately to severely impaired in rodent models of five distinctly different types of memory disorder: middle-aging, early-stage Huntington's Disease (HD), Fragile-X Syndrome (FXS), early-life stress, and menopause. Evidence obtained with newly introduced light microscopic techniques indicates that LTP-related reorganization of the spine cytoskeleton is defective in at least three of these cases. Infusions of Brain-Derived Neurotrophic Factor (BDNF) rescued LTP in four of the models and restored activity-driven changes to the cytoskeleton in two so far studied. Increasing brain concentrations of BDNF, using daily drug regimens developed as one of goals the previous PPG, produces similar effects. The proposed studies have the following objectives: i) test the specific prediction that activity-driven cytoskeletal reorganization is abnormal in the different rodent models of memory impairment (this entails adding a mouse model of Angelman's Syndrome to those used in previous work);ii) identify reasons (enzyme, signaling abnormalities) contributing to cytoskeletal defects in the different models;and iii) test if chronic up-regulation of BDNF increases BDNF signaling at synapses and restores actin signaling in spines. There will be four subprojects, directed by different PIs: each with its own rodent models and with different aspects of cytoskeletal signaling as a focus. A Core facility will make up the fifth subproject and will provide analytical, administrative, and animal services for the program. In all, the proposed studies are expected to test for the presence of a common neurobiological defect contributing to synaptic plasticity and memory disorders of different origin and to evaluate a clinically relevant strategy for normalizing spine plasticity and behavior. Project 1 Abstract (Christine M. Gall PI). In a number animal models of human cognitive impairment there are disturbances in activity-induced remodeling of the dendritic spine actin cytoskeleton and processes of long term potentiation (LTP) that depend upon it. Studies in this program have provided novel evidence that, for several models, Brain-derived neurotrophic factor (BDNF) can rescue both processes. These results suggest that processes of spine actin remodeling represent a final common path impacted in various conditions of cognitive dysfunction and that, through effects on this process BDNF might offset cognitive deficits of different origins. Project 1 will test the hypothesis that deficiencies associated with signaling in BDNF's TrkB receptor underlie LTP deficits in the Fmr1-knockout (KO) mouse model of Fragile-X Syndrome (FXS) (a mental retardation syndrome with susceptibility for autism spectrum disorder or ASD) and provide a first test of whether the LTP consolidation deficits exhibited by the Fmr1-KO mice are typical of other genetic disorders with high comorbidity for ASD. Specific Aim 1 will test the hypothesis that synaptic BDNF signaling through its TrkB receptor is regulated by neighboring receptor systems and enhanced by increased levels of endogenous BDNF. Subaims will test if the frequency sensitivity of TrkB activation depends on mGluR5 and NMDA receptors and if levels of TrkB activation are elevated with in vivo BDNF protein content is increased. Specific Aim 2 will identify deficits in TBS-induced signaling contributing to deficiencies in spine F-actin stabilization in Fmr1-KOs and, in particular, if there are impairments in the effects of theta burst afferent stimulation on signaling through TrkB and its downstream effectors Src, MAP kinase and the Rho-GTPase Rac1. Aim 3 will test if TBS-induced LTP and aspects of spine actin remodeling that are perturbed in Fmr1-KO mice, are similarly impaired in murine models of other autism associated disorders;to this end BTBR T+ tf/J mice will be evaluated. These studies will identify mechanisms underlying deficits in LTP stabilization in FXS model mice and therefore targets for therapeutics designed to improve cognitive function in autism associated disorders. Project 2 Abstract (Gary Lynch PI). The abnormal spine morphology found in disorders of memory and cognition is suggestive of defects in the local cytoskeleton. Evidence that signaling pathways controlling the organization of actin networks are disturbed in several instances of retardation accords with this idea. The proposed work uses recent advances in immunofluorescence microscopy and image reconstitution to test if activity-driven reorganization of the spine cytoskeleton is defective in rodent models of human conditions in which memory is impaired. The project involves two sets of experiments. Aim One will define signaling pathways used by normally present neuromodulatory factors [Brain-Derived Neurotrophic Factor (BDNF), estrogen] to potently influence the assembly and stabilization of actin filaments within spines following the induction of long-term potentiation (LTP). Changes in the availability of both BDNF and estrogen have been implicated in the failure of LTP to consolidate (stabilize) in the animal models. Aim Two will use the results from Aim One to identify LTP and learning-related defects in spine actin signaling in rodent models of middle-age, early stage Huntington's Disease (HD), and surgical menopause. Additional work addresses the prediction that BDNF will both normalize actin signaling in these cases and, for the HD model mice, will slow the progression of neuropathology in the neostriatum. The proposed work will substantially increase our understanding of how diverse memory disorders disrupt the final steps in consolidating synaptic changes and memory, and lay the groundwork for rigorous testing of a mechanism-based, pharmacologically plausible strategy for correcting such disruptions. Project 3 Abstract (Michel Baudry PI). The general model of LTP motivating the proposed PPG involves three, inter-related categories of events set in motion within dendritic spines by theta burst afferent stimulation (TBS): 1) proteolysis of key structural proteins and transmembrane receptors by calpain;2) reorganization and re-stabilization of the cytoskeletal network;and 3) local protein synthesis. According to the model, each of these elements is modulated by a number of agents, including Brain-Derived Neurotrophic Factor (BDNF), released during TBS. It is further proposed that disturbances to the model's components, or to the modulatory factors, are responsible for a broad array of memory and cognitive disorders. The first broad goal of SubProject 3 for the next two years is to test and expand the protein degradation argument of the general hypothesis. This will involve testing whether calpain activation is a necessary step for TBS-induced actin signaling/polymerization within dendritic spines. It will also involve testing the hypothesis that calpain activation is a consequence of ERK-mediated phosphorylation triggered by BDNF release. The second broad goal makes use of the results from the above experiments, and from other projects in the program, to investigate the causes and potential treatments for spine defects in a mouse model of Angelman Syndrome, a retardation disorder involving a ubiquitin ligase (E6-AP) targeting several elements intimately related to cytoskeletal reorganization. Specifically, Aim 3 will test if local protein synthesis and actin signaling are aberrant in the Ube3a knockout mouse model of Angelman Syndrome. In all, Project 3 addresses fundamental questions regarding spine mechanisms of synaptic plasticity and applies this knowledge to investigate possible causes of, and treatments for, synaptic and cognitive disorders associated with Angelman syndrome. Project 4 Abstract (Tallie Z. Baram, PI). Chronic stress during early postnatal life (ES) results in enduring deficits of learning and memory that become prominent during middle age, associated with profound disturbances in LTP and structural defects of apical dendrites and spines in hippocampal fields CA1 and CA3. ES persistently up-regulates the expression of the stress-activated neuropeptide CRH in hippocampus, and CRH damages spines and dendrites in domains that are impaired after ES, suggesting that CRH may be involved in an ES-provoked cascade of events that eventually leads to synaptic dysfunction. Selective disturbances to synaptic plasticity and dendrite/spine integrity are shared among ES and several disorders discussed elsewhere in this application, and preliminary work suggests that common mechanisms are involved. This project will test the hypothesis that, acting via CRH-CRH receptor signaling, ES leads to two types of disturbances of actin organization in dendritic spines: (1) ES interferes with basal actin polymerization resulting in spine loss;(2) In common with other disorders discussed in this project, ES deranges activity-driven assembly and stabilization of the spine actin-skeleton. Accordingly, the first aim of this 2 year ARRA-supported proposal is to test if actin stabilization mechanisms known to be defective in various rodent models of memory disorders are impacted by CRH-CRH-receptor signaling, and to define the responsible mechanisms. The second aim is to determine the role of endogenous CRH, which is pathologically elevated in ES graduates, in both activity-induced and basal derangements of actin dynamics in spines of middle-aged ES graduates. Collectively, these studies will significantly increase our understanding of why early-life stress leads to cognitive dysfunction in adulthood and set the stage for testing pharmacologically plausible strategies for ameliorating this clinically important neuropsychiatric problem. Project 5, Core, Abstract (C.M. Gall, PI). An advantage of conducting research within a coordinated Program Project is the ability to create core facilities to (i) efficiently and economically share resources and administration support, (ii) provide infrastructure for the scientific objectives of the program, (iii) provide mechanisms for disseminating results and facilitating discussions within the group, and (iv) coordinate interactions with outside investigators to obtain critical evaluation, technical advice and intellectual input to keep the work on track and at the cuing edge of technologies in the field. To this end an Analytical, Administrative and Animal Core (Core A) will be created and directed by Dr. Christine Gall. Core A will address 6 specific aims. Aim 1 is to establish a facility within the Core, for Deconvolution Microscopy-Image analysis, Electrophysiology, Behavioral Assays and BDNF Protein Assays to provide for shared analytical needs of the various subprojects, to ensure that data obtained within the program can be reliably compared across projects, and to make most the economical use of resources and technical support;this will include training and oversight of technical personnel. Facilities for Microscopy and Electrophysiology will be established in years 1 and 2, respectively. Aim 2 is to oversee animal purchase and maintenance costs, genotype mice for the program, and establish a video-monitored system for behavioral studies. Aim 3 is to provide ampakine drugs and neurotrophin reagents (BDNF peptides and antagonists) to subprojects;this includes contracting the synthesis of ampakines from outside sources. Aim 4 is to coordinate collaborations and integration of research results among subproject laboratories through meetings of all program investigators and smaller collaborative groups. Aim 5 is to coordinate interactions with outside investigators to provide review and oversight, technical advice, and outside intellectual input to program investigators: formal internal and external advisory board have been created to help meet these goals. Aim 6 is to provide administrative support and computer assistance for all program investigators. "